Sickle cell anemia (SSA) is a chronic, debilitating illness that leads to painful, vaso-occlusive crises and progressive organ failure. Knowledge of the factors which regulate the sickling process in the intact red cell will provide a greater understanding of the pathophysiology of sickling in vivo, and will provide methods for testing potential antisickling drugs. Nuclear magnetic resonance (NMR) spectroscopy is a powerful device for nondestructively obtaining biochemical information from intact cells. P-31 NMR spectroscopy will be used to relate intracellular pH, and 2,3-DPG -Hb binding to cell volume, Hb concentration and Hb - oxygen affinity in intact, normal and sickle cells. These relationships will first be explored in a model system in which the cell volumes are altered by adjustment of the osmolarity and solute composition of the cell suspension. This model will be compared to one in which red cell volumes have been increased by potential antisickling drugs that render cell membranes permeable to Na+ (tellurite, cetiedil), or decreased by other drugs that increase the permeability to K+ (valinomycin). Red cells from SSA patients will then be separated by means of discontinuous arabinogalactan gradient density centrifugation into subpopulations of succeedingly increasing density. Using P-31 NMR, the intracellular pH, 2,3-DPG concentration and the degree of 2,3-DPG - HbS binding for each subpopulation will be estimated, and compared to the values predicted by the model systems, based on cell volume, Hb concentration and Hb - oxygen affinity. These studies will test the hypotheses that the dense cells in SSA have lower pH as a result of progressive water loss and increasing HbS concentration, and that antisickling agents that act to decrease HbS concentration may provide additional antisickling action by raising intracellular pH. The average cell ages in these subpopulations will be estimated from pyruvate kinase activities, and C-13 NMR will be used to study rates of glycolytic metabolism. It will be determined whether glycolytic activity is a reflection of the younger average age of sickle cells, or whether this activity is altered by the sickling process. By performing these experiments on SSA patients both during vaso-occlusive crisis and during good health, it will be determined whether alterations in the physiologic properties of the sickle cell subpopulations can be related to the development of the vaso-occlusive state.